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目的:克隆结核分枝杆菌持续感染期抗原Rv1733c基因,构建其原核表达载体,并在大肠杆菌中进行表达和纯化。方法:采用聚合酶链反应(PCR)方法从结核分枝杆菌H37Rv基因组中扩增出Rv1733c基因片段,克隆入pMD18-T载体,序列测定正确后将其亚克隆入原核表达载体pPro-EXHTb,并在大肠杆菌DH5α中进行表达,表达蛋白经SDS-PAGE及Western-blot分析后,以Ni-NTA亲和层析纯化蛋白。结果:成功克隆了Rv1733c基因片段并构建了其原核表达载体pPro-EXHTb-1733c,转化E.ColiDH5α后能表达大小约30 KD的蛋白,Western-blot分析表明表达产物正确。通过亲和层析获得纯化蛋白。结论:成功构建结核分枝杆菌持续感染期抗原Rv1733c原核表达载体pPro-EXHTb-1733c,并获得纯化蛋白,为研究新型结核疫苗的靶抗原奠定了基础。
OBJECTIVE: To clone the Rv1733c antigen of Mycobacterium tuberculosis persistent infection and construct its prokaryotic expression vector, and express and purify it in E. coli. Methods: The Rv1733c gene fragment was amplified from the Mycobacterium tuberculosis H37Rv genome by polymerase chain reaction (PCR) and cloned into pMD18-T vector. After sequencing, it was subcloned into prokaryotic expression vector pPro-EXHTb The recombinant protein was expressed in Escherichia coli DH5α. The expressed protein was analyzed by SDS-PAGE and Western-blot. The protein was purified by Ni-NTA affinity chromatography. Results: The gene fragment of Rv1733c was successfully cloned and its prokaryotic expression vector pPro-EXHTb-1733c was constructed. After transformed into E. coli DH5α, the protein of about 30 KD was expressed. Western-blot analysis showed that the expression product was correct. Purified proteins were obtained by affinity chromatography. CONCLUSION: The prokaryotic expression vector pPro-EXHTb-1733c of Mycobacterium tuberculosis persistent infection antigen Rv1733c was successfully constructed and the purified protein was obtained, which laid the foundation for the study of target antigen of new tuberculosis vaccine.